The purpose of the proposed research is to characterize and to determine the physiological significance of a phosphorylation system (endogenous protein kinase and substrate) detected in the outer plasma membrane of 3T3 cells. The activity is correlated with cell growth in 3T3 cells: it is greater in growing cells than in quiescent ones; it decreases as cell density increases; it is 5-10 fold greater in SV40-3T3 cells than in normal 3T3. Stimulation of quiescent cells with serum brings about a 2-4 fold increase in incorporation of phosphate as does also treatment with small amounts of insulin, prostaglandin E1 or F2alpha. We plan to characterize the protein kinases and substrates in quiescent and growing 3T3 cells, in temperature-sensitive cell cycle mutants, and in several classes of SV40 transformed 3T3 cells. We will assay incorporation of 32P-phosphate from gamma32P-ATP into acid-precipitable protein of intact cells and isolated membrane preparations. In some experiments, cells will be labeled in vivo by growth in medium with 32P-phosphate. Double labeling procedures using 32P and 33P-phosphate or 32P-phosphate and 14C-amino acids will be used to compare cells in different growth states. We plan to use membrane extraction and vesiculation procedures combined with immunoprecipitation of the membrane proteins to concentrate and select the natural phosphorylated substrates. These proteins will be examined further by SDS polyacrylamide gel electrophoresis and radioautography.